Optimized Tangential Flow Filtration for Efficient Isolation of Highly Pure and Bioactive sEVs

Small
extracellular vesicles (sEVs) have demonstrated
significant
therapeutic potential. However, the lack of efficient and scalable
methods for sEV separation remains a critical barrier to their large-scale
production as therapeutic agents. Tangential flow filtration (TFF)
has emerged as a promising technique for large-scale sEV separation,
but the absence of comprehensive and systematic studies makes it challenging
to determine optimal TFF parameters for sEV isolation. In this study,
we investigated the effects of key TFF parameters, including shear
stress, molecular weight cutoff (MWCO), transmembrane pressure (TMP),
and washing–filtration cycles, on sEVs separation and impurity
removal. We evaluated particle concentration, size distribution, morphology,
chemical properties, fluorescence, and protein content to assess the
impact of these parameters. The results revealed that excessive shear
stress (>9.05 Pa) during TFF led to protein aggregation and sEV
damage.
Using a filter membrane with an MWCO of 750 kDa and maintaining TMP
between 3 and 5 psi prevented sEV leakage and achieved moderately
high filtration efficiency. Based on these findings, optimal TFF parameters
were identified. sEVs isolated using optimized TFF conditions (TFF-sEVs)
were then compared with sEVs isolated via ultracentrifugation (UC-sEVs).
TFF-sEVs exhibited comparable physical properties and purity to UC-sEVs
but demonstrated superior proliferation-promoting activity and a 3-fold
higher recovery rate. Quantitative proteomic analysis revealed that
TFF-sEVs were enriched with extracellular vesicle membrane proteins
and corona proteins, contributing to their enhanced bioactivity. In
summary, optimizing TFF parameters enables efficient purification
of sEVs, balancing purity, recovery, and bioactivity, paving the way
for scalable production of sEVs and accelerating the clinical translation
of sEV-based therapeutics.